Various publications, including patents, published applications and scholarly articles, are cited throughout the specification. Each of these publications is incorporated by reference herein, in its entirety. Citations not fully set forth within the specification may be found at the end of the specification.
Coffee aroma and flavor are key components in consumer preference for coffee varieties and brands. The characteristic aroma and flavor of coffee stems from a complex series of chemical reactions involving flavor precursors (Maillard reactions) that occur during the roasting of the bean. Flavor precursors include chemical compounds and biomolecules present in the green coffee bean. To date, over 800 chemicals and biomolecules have been identified as contributing to coffee flavor and aroma. (Flament, I., 2002 “Coffee Flavor Chemistry” J. Wiley U.K.). Because coffee consumers are becoming increasingly sophisticated, it is desirable to produce coffee with improved aroma and flavor in order to meet consumer preferences. Both aroma and flavor may be artificially imparted into coffee products through chemical means. See, for example, U.S. Pat. No. 4,072,761 (aroma) and U.S. Pat. No. 3,962,321 (flavor). However, to date, there is little information concerning the influence of natural coffee grain components such as polysaccharides, proteins, pigments, and lipids, on coffee aroma and flavor. One approach is to select varieties from the existing germplasm that have superior flavor characteristics. A disadvantage to this approach is that, frequently, the highest quality varieties also possess significant negative agronomics traits, such as poor yield and low resistance to diseases and environmental stresses. It is also possible to select new varieties from breeding trials in which varieties with different industrial and agronomic traits are crossed and their progeny are screened for both high quality and good agronomic performance. However, this latter approach is very time consuming, with one crossing experiment and selection over three growing seasons talking a minimum of 7-8 years. Thus, an alternative approach to enhancing coffee quality would be to use techniques of molecular biology to enhance those elements responsible for the flavor and aroma that are naturally found in the coffee bean, or to add aroma and flavor-enhancing elements that do not naturally occur in coffee beans. Genetic engineering is particularly suited to achieve these ends. For example, coffee proteins from different coffee species may be swapped. In the alternative, the expression of genes encoding naturally occurring coffee proteins that positively contribute to coffee flavor may be enhanced. Conversely, the expression of genes encoding naturally occurring coffee proteins that negatively contribute to coffee flavor may be suppressed.
Coffees from different varieties and origins exhibit significant flavor and aroma quality variations when the green grain samples are roasted and processed in the same manner. The quality differences are a manifestation of chemical and physical variations within the grain samples that result mainly from differences in growing and processing conditions, and also from differences in the genetic background of both the maternal plant and the grain. At the level of chemical composition, at least part of the flavor quality can be associated with variations in the levels of small metabolites, such as sugars, acids, phenolics, and caffeine found associated with grain from different varieties. It is accepted that there are other less well characterized flavor and flavor-precursor molecules. In addition, it is likely that structural variations within the grain also contribute to differences in coffee quality. One approach to finding new components in the coffee grain linked to coffee quality is to study the genes and proteins differentially expressed during the maturation of grain samples in different varieties that possess different quality characteristics. Similarly, genes and proteins that participate in the biosynthesis of flavor and flavor-precursor molecules may be studied.
Lignin is a phenolic polymeric material, which in angiosperms is primarily composed of three phenylpropanoid pathway-derived compounds: p-coumaroyl alcohol, coniferyl alcohol and sinapyl alcohol, i.e., the major monolignols found in plant (Hatfield R et al. 2001). These monolignols produce respectively p-hydroxyphenyl H, guaiacyl G, and syringyl S units when incorporated into the lignin polymer. Although exceptions exist, in a dicotyledonous angiosperm such as coffee, lignins consist principally of G and S units with traces of H units (Boerjan W et al. 2003). These complex polymers contribute compressive strength and increased water impermeability of the extracellular cell wall polysaccharide-protein matrix (Whetten R W et al. 1998). One response to pathogen ingression in plants is to increase the production of lignins in the cell wall, thereby reinforcing the cells surrounding the infection site and restricting further pathogen growth. (Vance C et al. 1980). Furthermore, other types of stresses, such as elevated levels of H2O2 and reduced cellulose synthesis, also result in an increased production of lignin, indicating that elevation of lignin synthesis is part of the more general stress response system in plants. (Wu G et al. 1997; and Logemann E et al. 1997).
The biosynthetic pathway for the monolignols has been controversial, with the model for the pathway changing several times in recent years (Dixon R A et al. 2001; and, Humphreys J M et al. 2002). The synthesis of lignin monomers, which is part of phenylpropanoid metabolism, begins with the deamination of phenylalanine, continues with successive hydroxylation and methylation reactions on the aromatic ring, and ends with the conversion of the side-chain carboxyl to an alcohol group (Boerjan et al. 2003). As shown in FIG. 1, the enzyme 4-hydroxycinnamoyl-CoA ligase (4CL) catalyzes an early reaction in the pathway to monolignol synthesis, the formation of the CoA esters caffeoyl-CoA, feruloyl CoA, and 5-hydroxy-feruloyl CoA (Lee et al. 1997). cDNA encoding this protein have recently been obtained and characterized from coffee, see WO 2007/044992, claiming priority to U.S. Provisional Application No. 60/726,298.
In angiosperms species, the first lignol specific enzyme identified was caffeic acid O-methyltransferase (COMT). COMT is capable of converting caffeic acid to ferulic acid, as well as converting 5-hydroxyferulic acid to sinapic acid. (Dixon et al. 2001). Down regulation of the COMT gene in maize (Zea mays) has been shown to cause a significant reduction of COMT activity (a fall of 70 to 85%), resulting in modification of lignin content and composition, and indicates that this enzyme is a key enzyme for lignin synthesis. (Piquemal J et al. 2002). Recently, the 2.2-Å crystal structure of an alfalfa COMT protein complexed with the cofactor SAH (S-adenosyl-L-homocysteine) and the substrate ferulic acid has been accomplished by Zubieta et al. (2002). This has allowed for the development of a model to explain the catalytic mechanism of COMT. This model indicates that the 3- or 5-hydroxyl group can be deprotonated by His269, facilitating the transfer of the reactive methyl group of SAM. The crystal structure of the alfalfa COMT also indicated specific residues that, a) interact in SAM recognition, b) are involved in substrate recognition, and c) are involved in various aspects of the catalytic reaction (Zubieta et al. 2002).
Ferulic acid generated by COMT can be hydroxylated by ferulate 5 hydroxylase (F5H), which is a cytochrome P450-dependent monooxygenase, to form 5-hydroxy-ferulic acid. F5H is also capable of hydroxylating coniferaldehyde and coniferyl alcohol forming 5-hydroxy-coniferaldehyde and 5-hydroxy-coniferyl alcohol respectively (Meyer K et al. 1996). F5H is believed to be potentially a rate limiting step in syringyl lignin biosynthesis, a proposal supported by the observation that an Arabidopsis mutant deficient in F5H expression is also affected at the level of sinapate esters accumulation in siliques and seeds (Ruegger M et al. 1999). All the products of F5H are also substrates for a second O-methylation catalyzed by COMT1 (FIG. 1).
CCoAOMT is a bifunctional enzyme which converts caffeoyl CoA to feruloyl CoA and 5-hydroxy-feruloyl CoA to sinapyl CoA (Inoue et al. 1998), and a CcOAOMT has been directly shown to be involved in lignin biosynthesis in the differential tracheary elements of Zinnia elegans (Ye et al. 1995). cDNA encoding CCoAOMT proteins have also been isolated and characterized from coffee, see WO 2007/044992, claiming priority to U.S. Provisional Application No. 60/726,298.
Another enzyme specifically involved in lignol biosynthesis is cinnamoyl-CoA reductase (CCR), and this enzyme catalyzes the conversion of feruloyl CoA and 5-hydroxy-feruloyl CoA into coniferaldehyde and 5-hydroxy-coniferaldehyde respectively, leading directly into the biosynthesis of G (coniferaldehyde) and S (5-hydroxy-coniferaldehyde) lignin units (Ma et al. 2005). In tobacco, down regulation of the CCR gene using an antisense construct generated plants with abnormal development and reduced growth, as well as abnormal leaf morphology and collapsed vessels. There was also an associated reduction in the level of G lignin compounds (Ralph J et al. 1998). One of the last enzymes involved in the monolignol pathway is cinnamyl alcohol dehydrogenase (CAD), which catalyzes the NADPH dependent conversion of coniferaldehyde, 5-hydroxy-coniferaldehyde and sinapaldehyde to the corresponding alcohols (Kim S J et al. 2004). In Arabidopsis, single mutants of the CAD genes AtCAD-C and AtCAD-D were found to have lower CAD activities, while a the double mutant obtained by crossing the two mutants had a 40% decrease in stem lignin content, demonstrating that these are the main CAD genes involved in stem lignin synthesis (Sibout R et al. 2005). This latter data indicates that altering a late step in lignol synthesis (i.e., altered CAD expression/activity), can be useful to influence the types of lignin generated, as well as the quantity of lignin formed.
There is little information in the literature concerning the levels of lignin in mature green coffee grain. Previously, it has been suggested that coffee grain had a lignin content of approximately 5% (Dart, S. and Nursten, H. 1985 Volatile components. In Coffee, Volumne 1; Chemistry, ed Clarke, R. and Macrae, R. Elsevier Applied Science, London, p 223-265). More recently, a compositional analysis of green grain has indicated that the carbohydrates, fat and protein made up 72% of the grain, leaving 28% of the grain corresponding to chlorogenic acids, minerals, lignin, amino acids, trigonelline, caffeine, and other compounds (Oosterveld, A., Harmsen, J., Voragen, A. and Schols, H. 2003 Extraction and characterization of polysaccharides from green and roasted C. arabica beans. Carbohydrate Polymers, 52, 285-296). From this latter data, it can be estimated that approximately 5-8% of the green grain is lignin. Other evidence for presence of significant amounts of lignin in the secondary cell walls of coffee grain cells was obtained by several different staining techniques and the use of light and transmission electron microscopy (Dentan, E. 1985. The microscopic structure of the coffee bean. In Coffee botany, biochemistry, and production of beans and beverage. Eds Clifford, M. and Willson, K. Croom Helm, London).
The lignin of the coffee grain is presumably involved in the maintenance of cellular structure, especially in the secondary cell walls of the grain, and likely also contributes to stress and insect resistance. In addition to being important for the overall health and structure of the coffee grain, it is likely that coffee grain quality can be influenced by the quantity, type, and structure of the lignin present. Lignin monomers and polymers may be directly involved in some of the chemical reactions that form coffee aromas/flavors and those that cause protein and polysaccharide degradation in the green coffee grain during coffee roasting. For example, lignin is believed to be a participant in the Maillard reaction, and potentially contributes to the generation of phenylpropanoid-derived aroma molecules such as guaiacol and 4-vinyl guaiacol. (Yeretzian C et al. 2002; and Logmann; Sagehashi, M. Miyasaka, N. Shishido, H., and Sakoda, A. 2005, Bioresource Technol. in press).
Lignins are also likely to be involved in melanoidin formation in coffee, and hence contribute to the overall antioxidant capability of this fraction. (Delgado-Andrade C et al. 2005). Lignin structure and/or quantity could also affect coffee quality indirectly by its influence on grain properties like water permeability and cell wall structure, thereby influencing, for example, the rate of water loss and the grain heating profile during coffee roasting, as well as the capability of the grain to trap volatile gases formed within the coffee endosperm during roasting (Yeretzian C et al. 2002).
Interestingly, it is believed that one or more coffee genes involved in lignin synthesis described herein is involved in the synthesis of coffee flavor molecules, or currently unknown flavor precursor molecules, in a similar fashion to that demonstrated recently in strawberry. Strawberries contain an unusual group of aroma compounds related to 2,5-dimethyl-3(2H)-furanone (DMMF). This particular compound is generated from 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) via an S-adenosyl-L-methionine dependent O-methyltransferase FaOMT with very high homology to the lignin synthesis enzyme COMT. The expression pattern of the FaOMT, and the enzymatic activity in the different stages of fruit ripening, suggests that FaOMT is also involved in lignin formation within the achenes and the vascular bundles of the expanding fruit, in addition to playing an important role in the biosynthesis of strawberry volatiles such as vanillin and DMMF (Weim et al. 2002).
Variety differences in lignin structure and/or quantity can also alter the extractability properties of the respective roasted grain. In maize, four brown mdrib (bm) mutants are known: bm1, which affects in CAD activity, bm2, which is associated with an over-expression of COMT, bin3a and 3b, which represent an insertion and a deletion in a COMT gene, respectively, and the bm4 mutant, which is affected in cell wall composition. Marita et al. (2003), showed that the double mutant bm1-bm2 had lower lignin content relative to the wild type. In addition, parallel reduction of esterified p-coumaroyl CoA was observed in all mutants. All observation were associated with alteration of cell wall degradability in the maize mutant (Marita J M et al. 2003).
Despite of the importance of lignin synthesis to the overall welfare of the coffee plant, as well as its probable impact on several aspects of coffee quality, at present there is no available information detailing lignin biosynthesis in coffee.
From the foregoing discussion, it will be appreciated that modulating lignin content in coffee grain by genetically modulating the production of the proteins responsible for lignin biosynthesis would be of great utility to enhance the aroma and flavor of coffee beverages and coffee products produced from such genetically engineered coffee beans. Modulating lignin content in the coffee plant also has implications for protecting the coffee plant and its fruit from pathogens, herbivores, and insects. Accordingly, a need exists to identify, isolate and utilize genes and enzymes from coffee that are involved in the biosynthesis of lignins. The present invention addresses this need.